We recently found a role for complement (C) in delayed-type hypersensitivity (DTH), and in contact sensitivity (CS). This led to our finding a role for B cells. Now we will investigate B cell subsets and Ig isotypes that are involved. The B-1 B cell subset likely plays a role by secreting IgM, that may activate C, possibly leading to local T cell recruitment, especially early after sensitization (1-4 days). We also will explore B-2 cell IgG2a and IgG2b C-activating antibodies, and B cell APC function. SPECIFIC AIM #1. To identify needed B cell subsets. FACS purified normal B-1 vs B-2 cells will be transferred into pan B-cell deficient JH-/- mice. To determine if reconstituting B cells act at the elicitation phase, we also will transfer immune B cell subsets into already sensitized JH-/- mice, and then challenge the skin. Radiation chimeras that express dominant B-1 vs B-2 cells, also will be used. SPECIFIC AIM #2. Determine properties of B cells in CS. If B-1 cells are preferentially involved, we will determine if they are Thy-1+, that may be induced via an early release of IL-4 from activated NK 1.1+ cells, and activated B-1 cells may possibly migrate from peritoneum to lymph nodes, and potentially have diverse V regions, and higher Ag affinity. SPECIFIC AIM #3. Determine how B cells function in CS. APC function will be explored in mutant mice with Ig+ B cells, but no secreted Ig. The possible involvement of IgM will be determined by employing mutant mice that just lack secreted IgM. If they are deficient in CS, we will attempt reconstitution with purified IgM mAb. More definitively, we will create transgenes in JH-/- of relevant specific Ig isotypes, to attempt CS-reconstitution. Finally, we will use anti-TNP IgM mutant hybridoma lines, to determine which amino acid residues in the Fc portion are essential for DTH. IN SUMMARY. Our experiments will determine the B cell subset(s) and Ig isotype(s) involved in DTH, and whether B cells act in the afferent, and/or the elicitation phase. Unique participation of B-1 cells is postulated, as one part of B cell involvement. These studies bring new ideas about antibodies acting in eliciting T cell immunity, and may uniquely connect innate immunity (C), with acquired T cell immunity (DTH), via linkage provided by natural immunity (B-1 cell IgM). Since DTH is central to in vivo T cell reactivity, these studies may be important to an indepth understanding of T cell diseases and immune resistance, and thus may have wide applicability to human health.